Researchers have developed a new imaging technology to study stem cells as
they proliferate and differentiate, according to a recent paper in Circulation
Research (1). The technology deals with limitations of previous
cellular imaging techniques and paves the way for imaging cell lines that
could be used safely in stem cell therapies, the authors say.

Both human embryonic stem cells (hESCs) and human induced pluripotent stem
cells (iPSCs) can differentiate into any somatic cell type, and they have
shown promise in cell replacement therapies. But "it is vital that we
be able to track these cells both in real time and over time" in order
to better understand their behavior and potential, said study author Joseph
Wu of Stanford School of Medicine in Palo Alto, CA.

To visualize cellular behavior, researchers insert reporter genes, such as
those for fluorescent proteins, into the genomes of the cells. These
reporter genes typically insert randomly into the genome.

Random integration of reporter genes is problematic for a few reasons. Some
cells have single copies of the reporter gene while others will have
multiple copies. And, depending on where the reporter is inserted,
expression can be unstable, and can even damage the cells.

Such issues "not only are severely detrimental to the health of the cell,
but also add confounding factors to the study of the modified cells,"
Wu said.

To get around this, Wu and his co-workers used a technology called human
genome editing to insert reporter genes into hESC and iPSC genomes. In
genome editing, an engineered zinc finger nuclease creates a double-stranded
break in the target DNA, and the reporter gene is then integrated at the
break.

The researchers selected an insertion site in a gene on chromosome 19 called
PPP1R12C, which had been proven inessential to the cells' behavior and
unlikely to affect transcription of other genes. They inserted three
reporter genes into this locus – for fluorescence, bioluminescence, and
positron emission tomography (PET) imaging.

Wu and his colleagues found that the edited stem cells remained pluripotent
and maintained reporter gene expression for up to two months. They imaged
the cells successfully bothin vitro and in vivo. They also differentiated
the cells into cardiomyocytes and epithelial cells – two cell types that
have shown promise in treating myocardial infarction – and found they were
indistinguishable from unmodified cells, suggesting they could potentially
be used clinically.

Editing stem cells is not without its technical challenges, however, said Wu.
Cell lines need to be grown from a single edited cell, which can be
difficult, and the entire process takes a month or two. (Random integration
of a reporter gene takes as little as a day.) But because of the limitations
of random integration, being able to insert reporters via genome editing is "a
very necessary tool," he added.